Journal: Theranostics

Article Title: Extracellular matrix proteins refine microenvironments for pancreatic organogenesis from induced pluripotent stem cell differentiation

doi: 10.7150/thno.104883

Figure Lengend Snippet: Enhancement of endocrine progenitor lineage specification from iPSCs under COL2 or COL5 stimulation. (A) A schematic diagram of a three-stage endocrine progenitor development protocol. Cells were differentiated on Matrigel (MG) as a control, or MG-collagen coated plates with indicated collagen concentration (30 and 80: 30 and 80 µg/mL). I~V denote five types of collagens. Cells were collected at the end of 3-stage differentiation, and the gene expression of PDX1 (B) and NKX6.1 (C) under different conditions were determined by real-time PCR. The expression of PDX1 and NKX6.1 were normalized to IMR90 cells (n = 3 biological replicates in each group, except n = 4 for PDX1 of control group; n = 4 for NKX6.1 of I 80 group). (D) Flow cytometric analysis of SOX17 expression on day 5. The large box shows the percentage of SOX17 + cells, whereas the small box indicates a higher expression level of SOX17 (SOX17 ++ ). (E) Comparison of percentage of SOX17 ++ cells in DE. Results are shown as mean ± SD (n = 3 biological replicates for each group). (F) The gene expression levels of ISL1 and FOXA2 on day 10 were determined and normalized to IMR90 cells (n = 4 for the control group, n = 3 for the COL2 and COL5 groups). (G) Flow cytometric analysis of PDX1 + /NKX6.1 + cells collected at the end of the 3-stage differentiation. (H) Comparison of average percentage of PDX1/NKX6.1 expressing cells at the end of the differentiation (n = 3 biological replicates for each group). (B, C, E, F, H) Results are shown as mean ± SD. *, p < 0.05; and **, p < 0.01. (I) Immunofluorescence micrographs of PDX1 and NKX6.1 expressing cells at the end of the 3-stage differentiation. Cells were counterstained with DAPI (blue). Scale bar: 100 μm.

Article Snippet: Cell culture: Human iPSC lines IMR90 and DF4 from WiCell Research Institute were used for the study.

Techniques: Control, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Comparison, Immunofluorescence

Journal: Theranostics

Article Title: Extracellular matrix proteins refine microenvironments for pancreatic organogenesis from induced pluripotent stem cell differentiation

doi: 10.7150/thno.104883

Figure Lengend Snippet: COL2 or COL5 augments endocrine progenitor development from iPSCs. iPSC IMR90 were differentiated on MG (control), or MG-COL2 or COL5 coated substrates with concentrations of 80 µg/mL COL2 or 40 µg/mL COL5. (A, B) Differentially expressed genes in the EP cells. The Venn diagrams show the number of the differentially expressed genes in the COL2 (A) and COL5 group (B). ( p < 0.05, fold change > 2 or < 0.5) (C) qRT-PCR and RNA-seq analyses of key EP marker expression in cells collected at the end of the 3-stage differentiation. The left y-axis represents the expression levels which were normalized to IMR90 cells. The right y-axis represents fragments per kilobase of exon per million mapped (FPKM) by RNA-seq. Results are from three independent experiments and shown as mean ± SD. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 compared to the MG group. ns: not significant; ND, not detectable. (D) RNA-seq identified upregulation of key signature genes in EP development in the COL2 or COL5 group. Results were from three independent experiments and shown as mean ± SD. ( p < 0.05 and fold change > 2) (E-F) The heatmap of the EP signature genes that were upregulated significantly in the COL2 (E) or COL5 (F) group ( p < 0.05 and fold change > 1.5).

Article Snippet: Cell culture: Human iPSC lines IMR90 and DF4 from WiCell Research Institute were used for the study.

Techniques: Control, Quantitative RT-PCR, RNA Sequencing Assay, Marker, Expressing

Journal: Theranostics

Article Title: Extracellular matrix proteins refine microenvironments for pancreatic organogenesis from induced pluripotent stem cell differentiation

doi: 10.7150/thno.104883

Figure Lengend Snippet: COL2 and COL5 activate the canonical WNT signaling pathway during iPSC-EP differentiation. (A-B) Gene cluster plots displaying enriched signaling pathways involved in significantly upregulated genes in cells collected under COL2 (A) or COL5 (B) stimulation. The collagen untreated iPSC differentiation served as a control. An inner circle presents the expression level of each gene shown as log fold change (log FC), whereas an outer circle depicts the signaling pathways that each gene involved in. (C) β-catenin translocation from the cytoplasm to the nucleus in the COL2 (C2), COL5 (C5), and the control (Con) groups. β-actin served as a loading control. (D) Relative β-catenin expression level after normalization to β-actin (n = 5). Results were from five independent experiments and shown as mean ± SD. *, p < 0.05; and ***, p < 0.001 compared to the control group; ns: not significant. (E) iPSCs were differentiated to EP on MG (control), MG-COL2 or COL5 coated plates. 10 nM of the WNT-C59 (C59) was supplemented to the differentiation media at Stages 2 and 3. The gene expression levels of ISL1, PDX1, and FOXA2 were detected by qRT-PCR and normalized to IMR90 cells (n = 4 biological replicates for each group). Results were shown as mean ± SD. *, p < 0.05; **, p < 0.01; and ***, p < 0.001. (F) The interplay between the WNT/β-catenin and COL2/COL5 cues for enhanced EP development, determined by Western blot analysis. (G) Relative β-catenin expression normalized to β-actin (n = 4). **: p < 0.01; ns: not significant.

Article Snippet: Cell culture: Human iPSC lines IMR90 and DF4 from WiCell Research Institute were used for the study.

Techniques: Control, Expressing, Translocation Assay, Quantitative RT-PCR, Western Blot